Marburg I mutant of factor VII activating protease (FSAP) as risk factor for arterial thrombosis

ABSTRACT

A novel arterial thrombosis risk factor comprising one or more of the identified mutants of coagulation factor VII-activating protease (FSAP) is described. In addition, diagnostic determination methods for detecting these mutants which are identified as risk factors are described.

This application is a continuation-in-part of application Ser. No.09/912,559 filed Jul. 26, 2001, now U.S. Pat. No. 6,831,167, whichclaims priority to German Application Nos. 100 36 641.4, filed Jul. 26,2000, 100 50 040.4, filed Oct. 10, 2000, 100 52 319.6 filed Oct. 21,2000, and 101 18 706.8, filed Apr. 12, 2001. This application alsoclaims priority to German Application Nos. DE 102 12 246.6, filed Mar.19, 2002; and DE DE 102 38 429.0, filed Aug. 16, 2002. All of theabove-listed applications, in their entirety, are incorporated herein byreference.

The invention relates to mutants of factor VII-activating protease(FSAP) and to reduced blood plasma levels of FSAP as indicators of anincreased risk for the development and progression of atherothrombosis(or arterial thrombosis) and of the pathophysiological sequelaeresulting therefrom.

Atherosclerosis is a pathological change in the arteries which isassociated inter alia with hardening, thickening and loss of elasticitythereof and is regarded as the main cause of myocardial infarction andstroke and of other disorders. Numerous exogenous and endogenous factorsare thought to be responsible for the initiation and progression ofatherosclerosis, for example hypertension, hyperlipidemia, diabetes,toxins, nicotine, excessive alcohol consumption and inflammations. Theseinfluences are referred to as risk factors. However, with the increasingnumber of studies and improved analytical methods, in recent yearsfurther risk factors for atherosclerosis and subsequent arterialthrombosis have been found.

Risk factors are investigated in epidemiological studies, as in theBruneck study which has become well known among specialists in thefield. One thousand inhabitants of Bruneck, Italy, were recruited forthis study in 1990. Ultrasound investigations of the carotid artery andanalyses of a number of blood parameters and questioning of the subjectsmade it possible to establish a broad database for further follow-up ofthe development and progression of atherosclerosis. These investigationswere continued on the same subjects and analyzed at 5-year intervals. Amodel of the development of atherosclerosis and its progression wasderived therefrom. As a first result of this study, a connection wasfound between the development of atherosclerosis and known, traditionalrisk factors such as the aforementioned hyperlipidemia and otherfactors. However, if the atherosclerotic plaque reaches such an extentthat the blood vessel is occluded by up to 40%, other risk factorsbecome important and may significantly influence the further progress ofthe atherosclerosis and the vascular occlusion. These factors include inparticular plasma proteins which intervene in hemostasis. A reducedcoagulation-inhibitory potential contributes to this, e.g. reducedantithrombin or protein C levels, or the so-called APC (activatedprotein C) resistance. A reduction in the fibrinolytic potential maytherefore have a crucial influence on the progression of the vascularocclusion, as is observed for example when the levels of lipoprotein (a)in the blood are raised.

To date, risk factors for venous occlusive disorders have beenidentified, such as APC resistance (Factor V Leiden). The extent towhich a given risk factor increases the risk for a condition may beexpressed as an “odds ratio.” The odds ratio for venous occlusivedisorders in heterozygotes for the APC resistance mutation has beenreported to be about 5 to 8, compared with subjects without APCresistance. For arterial occlusive disorders, on the other hand, thisrisk factor has been reported with low odds ratios of average about 2.

The plasma samples and DNAs available from the subjects in the Bruneckstudy were therefore investigated once again for the presence of otherrisk factors for atherosclerosis, directing particular attention atrecently found mutants of coagulation factor VII-activating protease(=FSAP), referred to hereinafter as the FSAP Marburg I and Marburg IImutations.

German patent application 199 03 693.4 discloses a protease which can beisolated from blood plasma and which is able to activate coagulationfactor VII. This protease is also referred to as factor seven-activatingprotease (FSAP) (or as PHBP or PHBSP, corresponding to plasma hyaluronicacid-binding (serine) protease). FSAP therefore has procoagulantproperties. A particular property of FSAP is that of activatingsingle-chain plasminogen activators, such as prourokinase orsingle-chain tissue plasminogen activator (sct-PA). However, alone or incombination with plasminogen activators, FSAP can also be usedcorrespondingly to assist fibrinolysis, for example, in cases ofthrombotic complications.

The test systems which are now available and are described in the Germanpatent applications 199 03 693.4 and 199 26 531.3 make it possible notonly to detect FSAP but also to quantify the FSAP antigen content anddetermine the activity thereof in plasma. The antigen determination ispreferably carried out by means of an ELISA test. On the other hand, theactivity can in principle be determined through activation ofprourokinase to urokinase and conversion of a chromogenic substrate withsubsequent extinction difference measurement.

German patent application 100 52 319.6 discloses the use of these testsystems in investigations on healthy blood donors in which 5 to 10% ofsubjects were identified as having an average FSAP antigen content but amarkedly reduced potential for activation of prourokinase. Since thisprobably also applied to the isolated, individual proteases, thecorresponding DNAs were analyzed for further investigation from bloodcells. It was surprisingly possible in this case to identify inparticular a mutation (single nucleotide polymorphism; SNP; G/A inposition 1601). This modification leads in the protein to a Gly to Gluamino acid exchange in position 511 of the mature protein or in aminoacid position 534 of the FSAP proenzyme including the signal peptide.This amino acid exchange results in FSAP losing the ability to activateprourokinase to urokinase or at least suffering a considerablediminution in activity. The aforementioned mutation, called FSAP MarburgI, has to date been found in all samples having an average antigencontent but a reduced activity in the formation of urokinase fromprourokinase. These results are described, for example, in U.S.application Ser. No. 09/912,559, to which this application claimspriority, and which is incorporated herein by reference.

For example, genomic DNA from the blood of two subjects with reducedactivity and from four subjects with normal prourokinase activity wasisolated, all exons amplified and then the FSAP DNA sequence wasdetermined using the PCR primers. The result is shown in Table 1. Atotal of 4 nucleotide positions in the coding region were polymorphic,i.e. at these positions two bases were detected simultaneously. It cantherefore be assumed that these cases are heterozygous, having one wildtype and one mutant allele. Two of these (at positions 183 and 957) arethird base exchanges that do not result in amino acid exchange. Theother two, which were found only in the DNA of the subjects with reducedprourokinase activity, lead to amino acid exchanges as depicted in Table1.

TABLE 1 DNA sequence at nucleotide positions* Subject No. ProUK activity183 957 1177 1601 S83182 T G G G 9689 normal T/C G G G 9690 normal T/C GG G 9704 normal T G G G 9706 normal T G G G 9714 reduced T G G/C G/A9715 reduced T G G/C G/A *where 1 is A of the start codon. Amino acid atposition* Subject NT*:183 NT:957 NT:1177 NT:1601 No. ProUK activityAA*:61 AA:319 AA:393 AA:534 S83182 His Lys Glu Gly 9689 normal His LysGlu Gly 9690 normal His Lys Glu Gly 9704 normal His Lys Glu Gly 9706normal His Lys Glu Gly 9714 reduced His Lys Glu/Gln Gly/Glu 9715 reducedHis Lys Glu/Gln Gly/Glu *NT-Nucleotide position; AA-amino acid position,where 1 is the methionine of the leader peptide.

In order to study the correlation of the two FSAP mutations with Reducedprourokinase activating potency, the DNA of further individuals wasSequenced at these positions. The result is summarized in Table 2. All 6subjects Having reduced prourokinase activating potency wereheterozygous at the nucleotide Position 1601 (Gly-Glu exchange at aminoacid 534), and four were additionally heterozygous at nucleotideposition 1177 (Glu-Gln exchange at amino acid 393). None of the 11subjects in total having normal prourokinase activating potency orprourokinase activating potency in the lower normal range had theabovementioned heterozygosities. This result suggests that the exchangein amino acid position 534 is causally linked to reduced prourokinaseactivity.

TABLE 2 DNA sequence at Subject nucleotide position No. FSAP antigenProUK activity 1177 1601 9714 Normal Low C/G A/G 9715 Normal Low C/G A/G9802 Normal Low C/G A/G 10032 Normal Low G A/G 10039 Normal Low C/G A/G10047 Normal Low G A/G 9698 Lower normal range Lower normal range G G9702 Lower normal range Lower normal range G G 9711 Lower normal rangeLower normal range G G 9712 Lower normal range Lower normal range G G10038 Lower normal range Lower normal range G G 9689 Normal Normal G G9690 Normal Normal G G 9704 Normal Normal G G 9706 Normal Normal G G9803 Normal Normal G G 10043 Normal Normal G G

Some embodiments of this invention therefore relate to anatherothrombosis risk factor that consists of a mutant of coagulationfactor VII-activating protease (FSAP). In some embodiments of thisinvention, the risk factor is a mutant in which the FSAP proenzyme,including the signal peptide, has a Gly/Glu exchange at amino acidposition 534. This mutation is herein called the Marburg I mutation.

The corresponding nucleotide sequence of the FSAP proenzyme includingthe signal peptide shows a G/A base exchange at nucleotide position1601. The sequences of the above FSAP species are provided in Table 3.

The Marburg I mutation is sometimes found together with a Glu/Glnexchange at position 393 (position 370 in the mature FSAP proteinwithout the leader sequence), resulting from a G to C mutation atposition 1177 in the nucleotide sequence corresponding to the FSAPproenzyme including the signal peptide. The Glu/Gln exchange at position393 is herein called the Marburg II mutation.

TABLE 3 NT:1177 NT:1601 SEQ ID NO. Description AA:393 AA:534 1 wild-typenucleotide G G 2 Marburg I nucleotide G A 3 Marburg II nucleotide C G 4Marburg I and II C A nucleotide 5 wild-type protein Glu Gly 6 Marburg Iprotein Glu Glu 7 Marburg II protein Gln Gly 8 Marburg I and II proteinGln Glu

A PCR test for the Marburg I and II mutations was established and usedto investigate the DNA of the subjects recruited for the Bruneck study.The FSAP Marburg I mutation was found in 4.5% of all the samplesanalyzed. These findings were then assessed using the individual datacollected during the study to assess the development and progression ofatherosclerosis.

Surprisingly, the Marburg I mutation correlates with an increased riskof developing arterial atherosclerosis. An odds ratio of about “6.6” wascalculated for this FSAP mutation, i.e. a risk on the arterial side thatis comparable with the risk of APC resistance in the venous region. Inthis study the Marburg I polymorphism was the risk factor with thehighest odds ratio of all factors investigated, as shown in table 4. Itis particularly surprising that this mutation represents an independentrisk factor, making its own, marked contribution to the development andprogression of atherosclerosis after allowance for all previously knownrisk factors. This realization and the determination of the sequencescorresponding to the FSAP Marburg I mutation may therefore improve theprospects of diagnosing and treating heart diseases and vasculardisorders caused by atherosclerosis.

Atherothrombosis frequently leads, for example, to coronary arterydisease followed by myocardial infarctions. Depending on the vesselsaffected, the organ supplied thereby becomes involved. In the case ofthe carotid artery, this results in the brain being undersupplied withnutrients and oxygen and may, in the worst case, lead to a stroke. Otherorgans affected by atherothrombosis and subject to the risk of vascularocclusive disease and the sequelae resulting thereform are also affectedby the FSAP Marburg I mutant. This may result, for example, in disordersof the kidneys, liver, lungs, and other disorders.

Thus, the risk factor of this invention may indicate a geneticpredisposition to arterial thrombosis, and/or a genetic predispositionto the development of thromboses. The risk factor may also indicate agenetic predisposition to the development of atherosclerotic disordersand their sequelae, such as coronary artery disease, acute myocardialinfarction, pulmonary embolism, peripheral artery occlusion, acuteischemic stroke, and thromboembolism, in addition to arterialthrombosis. The risk factor may also indicate a genetic predispositionto thrombotic disorders and their sequelae, such as arterial and venousthrombosis, deep vein thrombosis, acute myocardial infarction, pulmonaryembolism, peripheral artery occlusion, acute ischemic stroke, andthromboembolism. The risk factor may also indicate a geneticpredisposition to at least one of arterial and venous occlusivedisorders, to at least one of atherosclerotic and thromboticrestrictions of organ functions, as well as to one or more of anginapectoris, myocardial infarction, and strokes.

Methods for examining the structure, sequence, and activity of the FSAPMarburg I and II mutants are described in the aforementioned Germanpatent applications, in particular in German patent application 1090 52319.6, as well as in U.S. application Ser. No. 09/912,559, all of whichare incorporated herein by reference. These methods include measurementof the FSAP protease activity, preferably in combination with an FSAPantigen test, and determination of the nucleotide sequence in themutated region by suitable test systems.

The arterial thrombosis risk factor of the invention may thus be definedas one or more FSAP mutants that have lost the ability to activatesingle-chain plasminogen activators or for which this ability is atleast impaired. In some embodiments, the risk factor is characterized byan FSAP mutant that has lost the ability to activate prourokinase or forwhich this ability is reduced.

A genetic predisposition to the development of arterial thrombosis canthus be identified by detecting one or more of the aforementioned FSAPmutants. Detection, as used herein, means determining if a mutant FSAPis present in a sample. Detection may be carried out by a variety ofmethods, as described below.

Detection of these FSAP mutants also indicates the predisposition to thedevelopment of arterial thromboses and the predisposition to thedevelopment of atherosclerotic or thrombotic disorders and theirsequelae, such as arterial and venous occlusive disorders, coronaryartery disease, acute myocardial infarction, pulmonary embolism,peripheral artery occlusion, acute ischemic stroke, deep veinthrombosis, and thromboembolism. The predisposition to development ofatherosclerotic or thrombotic restrictions of organ functions is afrequent cause of angina pectoris, myocardial infarction or strokes. Itis typical of all cases that the potential for activation ofsingle-chain plasminogen activators, such as single-chain tissueplasminogen activator (sc-tPA), and single-chain urinary plasminogenactivator (sc-uPA), or prourokinase, is reduced. The reduction of thisactivation potential can be detected in the blood but especially in theplasma.

In view of the great importance of FSAP mutants as atherothrombosis orarterial thrombosis risk factors, diagnostic methods for detecting themare very important. They may be based on determining at least one of areduced FSAP antigen concentration and a reduced activity of FSAP in thebody fluids of an individual. This may entail determination of thepotential for activation of single-chain plasminogen activators, such asprourokinase, in the body fluids.

In the context of the present invention, an individual, or donor, may bea mammal, such as a human. Relevant body fluids include whole blood,blood plasma, serum, as well as lymphatic, cerebrospinal, pleural,pericardial, peritoneal, and synovial fluids, tears, seminal plasma, andcell lysates.

Some embodiments of the invention include detecting heterozygous orhomozygous mutants of the FSAP proenzyme gene with a G/A base exchangeat nucleotide position 1601 by analysing the genomic DNA of anindividual, or the mRNA or cDNA derived therefrom. Some embodiments ofthe invention include detecting heterozygous or homozygous mutants ofthe FSAP proenzyme gene with a G/C base exchange at position 1177. Insome embodiments, both of these base exchanges may be detected.

In other embodiments, FSAP mutants may be detected at the protein level.Specific monoclonal or polyclonal antibodies may be used for thispurpose, as well as their corresponding Fab or F(ab′)₂ fragments.Histological investigation methods on tissues or in solutions extractedfrom tissues are also available. For examples of relevant tissues in thecontext of this invention, see table 4 below.

Exemplary antibodies are those specific for one or more of wild-typeFSAP, including its proenzyme with or without the signal sequence, andits fragments; and FSAP mutants comprising at least one of a Glu to Glnexchange at amino acid position 393 and a Gly to Glu exchange at aminoacid position 534, as well as their proenzymes with or without thesignal sequence, and their fragments. Antibodies herein thatspecifically recognize both wild-type and mutant FSAP sequences, such assequences corresponding to full-length active enzymes, proenzymes andenzyme fragments, are termed “FSAP-specific,” while those that arespecific for only wild-type or only mutant FSAP sequences are termed“wild-type FSAP-specific” and “mutant FSAP-specific,” respectively.

In some embodiments, the diagnostic method includes incubating a samplethat might contain FSAP mutant(s) with a first antibody immobilized on asolid support, and, after washing, adding a second, labelled antibody,washing again and measuring the signal elicited by the second antibody,wherein the second, labelled antibody may be a wild-type FSAP-specificantibody.

Another method comprises incubating a sample that might contain FSAPmutant(s) with a first, wild-type FSAP-specific antibody, immobilized ona solid support, and, after washing, adding a second, labelled antibody,washing again and measuring the signal elicited by the second antibody.

A further method comprises immobilizing the sample that might containFSAP mutant(s) on a support, adding a labelled antibody, alone or mixedwith an unlabelled antibody, and detecting the labelled antibody.

Yet another method comprises mixing an antibody immobilized on a supportwith the sample that might contain FSAP mutant(s) in the presence of alabelled FSAP mutant, and measuring the signal elicited by the label.

In the methods described above, the first or second antibodies, unlessstated otherwise, may include FSAP-specific, wild-type FSAP-specific, ormutant FSAP-specific antibodies.

An example diagnostic method in which the activity of FSAP is measuredcomprises incubating an FSAP-containing sample on a solid support ontowhich an FSAP-specific antibody has previously been coupled, and then,after washing out the free support, incubating the FSAP immobilizedthereon with reagents that allow determination of FSAP activity.

Some embodiments include diagnostic methods in which antibodies are usedto detect FSAP mutants by Western blotting for immunohistology,fluorescence-activated cell sorting (FACS), or comparable methods.

The diagnostic methods of the invention may also be carried out by theELISA technique. This entails binding an FSAP and/or FSAP mutant to amatrix, for example to a microtiter plate. For optimal presentation ofthe FSAP and/or FSAP mutant, it is possible to coat the plate withmonoclonal or polyclonal antibodies, or the F(ab′)₂ or Fab fragmentsthereof, before loading with FSAP and/or FSAP mutants. Since FSAP andFSAP mutants generally bind very well to dextran sulfate, heparin andsimilar substances, coating with these agents is also possible prior toloading with FSAP and/or FSAP mutants. After the support or microtiterplate has been washed, it is blocked where appropriate with the agentsknown for this purpose, such as detergent or albumin, washed and thenincubated with the solution to be tested. The solutions containingFSAP-specific antibodies include blood serum, plasma, and other bodyfluids, as well as synovial fluids, cerebrospinal fluid, saliva, tears,seminal plasma or cell lysates.

Incubation and washing of the support is followed by use of a suitabledetection reagent. The test substances required for detecting thevarious antibody classes, such as IgG, IgM, IgA, IgE, and the relevantsubclasses, are commercially available as labelled reagents. Detectionand quantification of the antibody titer can then take place by aphotometric examination, for example, by measurement of the extinctionbrought about by cleavage of a chromogenic substrate by an enzymecoupled to the anti-human antibody. It is also possible to measure thefluorescence emitted by a fluorescent group connected to the antibodyused for the detection. In addition, it is possible to carry out thedetection with a radiometric measurement, if the substance used fordetection is labelled with a radioactive group. Diagnostic methods inwhich the bound human antibodies are incubated with a labelledanti-human immunoglobulin or fragments thereof, or labelled protein A orprotein G, and in which the signal emitted by the bound, labelledsubstance is determined, have already proved very suitable in manyinstances.

It is also possible to detect the antibodies by a photometricmeasurement of the extinction caused by cleavage of a suitablechromogenic or fluorogenic substrate by enzyme-coupled anti-humanantibodies or fragments thereof, or protein A or protein G. Diagnosticmethods in which antibodies are detected by measuring the fluorescencecaused by a bound substance labelled with fluorescent groups are alsosuitable.

In some embodiments, monoclonal antibodies were prepared andcharacterized as follows:

Immunization

Three female balb/c mice (approx. 6 weeks old) were immunized with FSAP.The first injection consisted of 0.2 ml of the antigen (10 μg) mixedwith 0.2 ml of complete Freund's adjuvant. In the three following boostinjections (each 2 weeks apart) the antigen (20 μg in 0.2 ml) wasadministered without adjuvant (all injections i.p.). The immunogen wasdiluted in PBS. After the last injection, the serum titer was determinedby means of indirect ELISA by coating a microtiter plate with FSAP. Themouse with the highest serum titer was selected for the fusion.

Fusion

About three weeks after the last application, the antigen wasadministered on three successive days (10 μg in 0.1 ml i.v.). On thenext day (day 4) the mouse was sacrificed after taking blood. The spleenwas removed and the spleen cells were isolated. The spleen cells werethen fused with the murine myeloma cell line SP2/0-Ag 14. The fusionreagent was polyethylene glycol 4000 (Merck). The fusion was carried outusing a modification of the original Köhler/Milstein method. The cellswere distributed on 24-well culture plates. The medium used was Dulbeccomod. Eagle's medium with 10% fetal calf serum and HAT for selection.After about two weeks, the cell clones grown were transferred to thewells of a 48 well plate and coded.

Hybridoma Screening

The culture supernatant was taken from 1728 grown clones and assayed bymeans of ELISA for the presence of mouse IgG. With the aid ofimmobilized FSAP, mouse IgG-positive supernatants were tested forspecificity (ELISA). Of the cell lines assayed, 108 cell lines wereidentified as specific for FVII activator and stored in the frozenstate.

The two hybridoma cell lines denoted DSM ACC2453 and DSM ACC2454 wereselected for further studies. These cell lines were deposited on Apr. 5,2000, with the DSMZ—Deutsche Sammlung Von Mikroorganismen undZellkulturen GmbH, a depository which is subject to the Treaty ofBudapest regulations. The specificity of the antibodies produced by saidcell lines was confirmed by BIACORE and binding kinetics weredetermined. The two monoclonal antibodies are of the IgG1 type.

With the aid of the described antibodies against FSAP wild type andagainst its mutants, it is possible to carry out diagnostic methods fordetecting the mutants by:

-   -   a) incubating a sample that could contain one or more FSAP        mutants with a first antibody, immobilized on a solid support,        then, after washing, adding a second, labeled antibody and,        after washing out again, measuring the signal produced by the        second antibody, wherein the second antibody may comprise a        wild-type FSAP-specific antibody; or    -   b) incubating a sample that could contain one or more FSAP        mutants with a first antibody immobilized on a solid support,        then, after washing, adding a second, labeled antibody and,        after washing out again, measuring the signal produced by the        second antibody, wherein the first antibody is a wild-type        FSAP-specific antibody; or    -   c) immobilizing a sample that could contain one or more FSAP        mutants on a support and detecting the sample with a labeled        antibody, alone or in a mixture with an unlabelled antibody; or    -   d) incubating a sample that could contain one or more FSAP        mutants with an antibody immobilized on a support in the        presence of a labeled FSAP mutant, and measuring the signal        produced by the label.

Antibody fragments, such as Fab and F(ab′)₂ fragments, may also be usedin the diagnostic methods in some embodiments.

An example diagnostic method in which the FSAP activity is measured mayinclude incubating the protease-containing sample with a solid supportto which at least one of an FSAP-specific antibody, a wild-typeFSAP-specific antibody, or a mutant FSAP-specific antibody has beencoupled beforehand and, after washing out the solid support, incubatingthe FSAP fixed to the support with reagents which allow determination ofits activity.

In this connection, FSAP activity, such as protease activity, can bemeasured by photometric determination of the extinction appearingfollowing the action on chromogenic substrates.

It is also possible to determine FSAP protease activity by measuring:

-   -   its action of inactivating blood clotting factors VIII/VIIIa or        V/Va or    -   its action of shortening blood clotting times in global clotting        assays or    -   its action of activating plasminogen activators or    -   its action of activating blood clotting factor VII.

In some embodiments, FSAP action of activating plasminogen activatorsmay be measured, by examining FSAP activation of the

-   -   single-chain urokinase (scuPA, single chain urokinase        plasminogen activator) or the    -   single-chain-tPA (sctPA, single chain tissue plasminogen        activator).

The mutations responsible for the reduction of prourokinase activatingpotency can be detected at the DNA and RNA level by using methods thatare also used for detecting single nucleotide polymorphisms, for example

-   -   cDNA amplification of mRNA or amplification of the genomic DNA        and sequencing;    -   detection of the mutation at the cDNA level or genomic DNA level        or their amplification by    -   hybridization with sequence-specific probes which may also carry        labels for the detection, such as enzymes, alkaline phosphatase,        HRP and their substrates, fluorescent dyes, also        reporter-quencher pairs (such as, for example, scorpions,        molecular beacons, TaqMan probes), radioisotopes, chromophores,        chemiluminescence labels and electrochemiluminescence labels) or    -   methods such as selective 2′-amine acylation, electrochemical        oxidation of nucleic acids by “minor groove binder”        oligonucleotide conjugates, or by HPLC.

On the basis of the test results which were obtained by theabovementioned antigen assays and activity assays it was possible tostudy three groups of healthy donors regarding potential mutations atthe genomic level. For this purpose, blood was taken from the donors andthe blood cells were separated from the plasma by centrifugation. Theplasmas were then used to quantify the FSAP antigen and activity levelsand were divided according to the latter into three groups, namely into“normal/normal,” “lower normal range/lower normal range” and“normal/low.” The blood cells obtained were then used to extract genomicDNA and determine the FSAP genotype. The results depicted above in Table2 were determined.

Based on these results, it is now possible to detect rapidly one or bothof the mutants described, whether their genotype is heterozygous orhomozygous, at the level of the corresponding FSAP nucleotide sequence.Whereas the abovementioned antigen and activity assays reflected quitewell the genotype in a healthy donor, this can become difficult orimpossible when the FSAP plasma levels are influenced. Thus, parameterssuch as hormonal fluctuations, lifestyle, etc., as well as pathologicalconditions, may strongly influence antigen and/or activity levels. Asdescribed in the German Patent application 199 26 531.3, the measurableFSAP activity during a heart attack can increase markedly compared withthe normal value with scarcely increased antigen content. As a result,donors that have a reduced FSAP activity when healthy, now appear to be“average.”

For example, studies on whether patients with FSAP mutations run anincreased risk of suffering thrombotic complications such as heartattacks are possible only with difficulty, owing to the abovementionedrestrictions. On the other hand, for example, liver failures may lead toreduced plasma levels, and this likewise may lead to misinterpretationsof the “true” genetic predisposition. In contrast, a FSAP mutation assayat the DNA and/or RNA level is independent of temporary events. Thecombination of all of the assays mentioned allows a complete picture ofthe donor/patient, i.e. the evaluation of a potential mutation and ofthe acute state regarding an influence on the antigen-activity ratio.This may result in prophylactic and therapeutic measures.

As described above, heterozygous individuals whose blood plasma containsnormal FSAP at about 50% and the FSAP mutant at about 50% have beenfound. This results in an about 50% reduced activity level of plasmas inwhich both types of FSAP molecules are present. A very small proportionof individuals was found to be homozygous, their blood plasma containingthe FSAP mutant at 100%, in which the prourokinase activation potencywas virtually abolished. Plasma pools which have been obtained from theblood of 100 and more donors therefore also contain 5 to 10% of FSAPmutants, depending on the population. This results in a correspondingprobability to receive, in blood transfusions, donor blood plasma thatcontains an FSAP mutant. If blood containing an FSAP mutant isadministered to a recipient who cannot produce the mutant, the mutantmay be recognized as extraneous and appropriate antibodies can begenerated. Subsequent administration of the FSAP mutant at a later stagemay lead to immunological reactions in the recipient, the side effectsof which are familiar to the skilled worker.

Conversely, in a homozygous blood recipient who produces only an FSAPmutant but not normal FSAP, the latter is recognized as “extraneous” andthe appropriate antibodies against it are produced.

FSAP affects hemostasis and the cellular processes connected therewith.By involvement in blood clotting and/or fibrinolysis, it also affectsthe wound healing reaction. Moreover, FSAP, due to its property ofhaving a high affinity to glycosaminoglycans, can bind to cells andother matrices and therefore is probably physiologically andpathophysiologically involved in cell migration and cellular-proteolyticprocesses.

FSAP-specific antibodies thus may influence all FSAP-mediatedactivities. In the case of autoantibodies against FSAP appearing, it ispossible that, in addition to an impairment of the physiologicalfunctions, immunocomplexes (FSAP+antibody) contribute to side effects ofknown autoimmune diseases. This may lead, for example, to vasculitideslocally in the endothelium. Neutralization of FSAP activity as aprofibrinolytic agent could also contribute to a thrombosis-promotingstate.

There is, therefore, a need for a diagnostic method for detecting theabove described antibodies.

Some embodiments of the invention, therefore, relate to diagnosticmethods for detecting antibodies against factor VII-activating protease(FSAP) and/or against one or more FSAP mutants formed by the exchange ofone or more amino acids. The method may comprise mixing a sample whichcould contain antibodies reactive with the FSAP and/or FSAP mutantsfixed to a solid support, incubating, and, after washing, detecting theantibody bound to the FSAP(s) with a labeled human anti-immunoglobulinor a labeled protein A and determining the signal emitted by the boundlabeled substance.

This diagnostic method may also be carried out using the ELISA techniquein which FSAP and/or one or more FSAP mutants are bound to a matrix, forexample, to a microtiter plate. For optimal presentation of the FSAPand/or FSAP mutants, the plate may be coated beforehand with monoclonalor polyclonal antibodies or their F(ab′)₂ or Fab fragments prior toloading the plate with the FSAP and/or FSAP mutants. Since FSAP and itsmutants generally bind very well to dextran sulfate, heparin and similarsubstances, prior coating with these agents before FSAP binding is alsopossible. After washing, the support or the microtiter plate mayadditionally be blocked and washed using agents known for this purpose,such as detergent or albumin, and then incubated with the solution to beassayed. FSAP antibody-containing solutions may include blood serum,plasma and other body fluids, such as synovial fluids, CSF, sputum,tears, and seminal plasma, as well as cell lysates.

After incubating and washing the support, a suitable detection agent isthen used. The assay substances necessary for detecting the variousantibody classes such as IgG, IgM, IgA, IgE and the subclasses belongingthereto, are commercially available as labeled reagents. The antibodytiter may be detected and quantified by a photometric determinationmeasuring the extinction caused by cleavage of a chromogenic substrateby an enzyme coupled to the anti-human antibody. It is also possible tomeasure fluorescence emitted by a fluorescent group linked to anantibody used for detection. It is also possible to carry out thedetection using radiometric measurement, if the substance used fordetection is labeled with a radioactive group.

The determination of antibodies against FSAP and/or in particular FSAPmutants makes it possible to identify the risk involved in a bloodtransfusion prior to carrying out the transfusion and to avoid dangerouscomplications by suitable measures.

Some embodiments of the invention relate to a diagnostic method forimmunohistochemical detection of the blood clotting factorVII-activating protease (FSAP), its proenzyme, its mutants, or itsfragments. The method may comprise letting an FSAP-specific, labeled,monoclonal or polyclonal antibody, or one of its fragments, react with atissue sample, washing out the unbound antibody or its fragments, anddetermining the signal emitted from the bound antibody or one of itsfragments.

The method may also be carried out by letting an unlabelled monoclonalor polyclonal antibody or antibody fragment, directed against FSAP, itsproenzyme, its mutants, or its fragments, react with a tissue sample,washing out the unbound antibody or its fragments, then letting alabeled anti-antibody or its fragments react with the tissue sample,and, after washing out the unbound labeled anti-antibody, determiningthe signal emitted from the bound anti-antibody or its fragments.

It was also found that monoclonal or polyclonal antibodies directedagainst FSAP are very well suited to detecting FSAP in tissue sectionsof human origin, when the antibodies are labeled with chromophoric orluminescent groups. FSAP-specific polyclonal antibodies obtained byimmunization of rabbits, sheep, goats, or other mammals are suitable forthe detection as well as monoclonal antibodies. Particularly suitablefor the histological specific detection of FSAP which may be presentboth in the active form and in the proenzyme form, as well as in afragment, are the monoclonal antibodies of hybridoma cell lines DSM ACC2453 and DSM ACC 2454. Complexes of activated FSAP with inhibitors suchas antiplasmin may also be detected in this way. Suitable for thispurpose are all common histological detection methods such as lightmicroscopy, fluorescence microscopy and electron microscopy.

Suitable for detecting FSAP in the abovementioned methods are both thecomplete polyclonal and monoclonal antibodies and their fragments suchas F(ab′)₂ or Fab, as long as they are labeled with a detectable group.The abovementioned antibodies or their fragments may be applied alone oras a mixture. This is particularly recommended in case one of therecognized epitopes is obscured. For example, a protein domain may notbe accessible for an antibody due to cellular association, but is boundby another antibody having specificity for a different FSAP region.Antibodies which are directed against human FSAP, such as against one ormore of the wild type and mutants of human FSAP, and which are describedin more detail in the German Patent application 100 52 319.6 may also beemployed for detection of FSAP in tissue sections of human origin.

The findings obtained so far on the immunohistochemical detection ofFSAP can be summarized as follows:

-   -   FSAP is detected in almost all of the human tissues studied up        until now;    -   endocrinologically active cells such as Leydig cells or the        endocrinologically active cells of the islets of Langerhans of        the pancreas are very strongly stained intracytoplasmatically        using antibodies against FSAP carrying chromophoric groups;    -   epithelia and endothelia display according to their location a        more or less strong intracytoplasmatic immunoreaction with        antibodies against FSAP;    -   gangliocytes and dendrites of the cortex display high        concentrations of FSAP, and this is detected by a strong        immunohistological color reaction with chromophoric antibodies;    -   plasma cells display an intensive intracytoplasmatic coloration        with chromophoric antibodies against FSAP;    -   mesenchymal stroma cells display in complex tissues only a weak        or no color reaction toward FSAP.

FSAP is thus a protein that can be regarded as a normal cellconstituent. So far FSAP was found located both intracellularly andextracellularly, with the former compartment being markedly morestainable. The inventive detection of FSAP by the mentioned antibodiesor their fragments makes it possible to identify the followingpathological processes:

-   -   endocrinologically active tumors and neuro-endocrine tumors;    -   angiogenic endothelia and endothelia of the capillary        endothelium; and also    -   angiogenically active tumors such as gliomas and glioblastomas,        but also, for example, vascular tumors such as        hemangioendothelioma or hemangiopericytoma and angiosarcoma;    -   wound healing reactions, granulation tissue and collagenoses;    -   atherosclerotic, (micro)thrombosed and necrotic areas;    -   neurodegenerative disorders such as Alzheimer's disease,        Parkinson's disease or as spongiform encephalitides, for example        caused by prion proteins;    -   gammopathies and myelomas.

FSAP may be detected by using monoclonal antibodies of hybridoma celllines DSM ACC 2453 or DSM ACC 2454.

The diagnostic method of the invention is illustrated in more detail bythe following example.

EXAMPLE I

The immunohistochemical reactivity of the FSAP-specific monoclonalantibodies of hybridoma cell lines DSM ACC 2453 and DSM ACC 2454 wasstudied by preparing from adult human tissue and malignant urologicaltumors 10 μm thick paraffin sections and subsequently de-waxing thesections which were treated in citrate buffer in the microwave for 3times 5 minutes. First, an unlabelled antibody of the abovementionedhybridoma cell lines was allowed to react with the sections for 30minutes. After washing out the tissue section, a labeled anti-mousedetection antibody was allowed to react with the tissue likewise for 30minutes and then the bound FSAP antibody was made visible by forming theAPAAP complex (Alkaline Phosphatase/Anti-Alkaline Phosphatase complex)and by staining with chromogen and counterstaining with hemalum.

As a negative control, each tissue was separately incubated with thedetection antibody—without prior incubation with the FSAP antibody—inorder to make potential unspecific reactions of the detection visible.In addition an antibody against α-keratin was included as a positivecontrol.

The results of the immunohistochemical study of normal human tissue aresummarized in Table 4.

TABLE 4 Antibodies against FSAP, clone DSMZ ACC2454 and DSMZ ACC 2453Human normal tissue 2454 2453 2454 2453 Esophagus Appendix Squamousepithelium 2+ 2+ Epithelia 1+ 3+ Secretory units 0   0   Musculature 1+1+ Acinar ducts 2+ 2+ Lymphatic follicle 1+ 2+ Musculature 1+ 1+ Plasmacells 2+ 3+ Stroma 1+ 1+ − 2+ Vascular endothelium 1+ 1+ Vascularendothelium 2+ 2+ Pancreas Cardia (stomach) Epithelia 1+ 2+ Foveolarepithelium 0   0   Islets of Langerhans 3+ 1+ Glandulae cardiacae 1+ 2+Duct epithelium 2+ 2+ Mucous secretory units 0   0   Vascularendothelium 1+ 1+ Oxyntic glands 3+ 3+ Salivary gland Musculature 1+ 1+Mucous end units 0   0   Vascular endothelium 1+ 1+ Serous end units 1+1+ − 2+ Corpus (stomach) Acinar ducts 1+ 1+ Foveolar epithelium 0   0 −1+ Striate ducts 1+ 1+ Corpus gland body 2+ 2+ Vascular endothelium 0 −1+ 0 − 1+ Musculature 0   1+ Liver Vascuiar endothelium 1+ 1+Hepatocytes 2+ 2+ Duodenum Bile ducts 0   0   Epithelia 0 − 1+ 1+Vascular endothelium 1+ (1+) Brunner's glands 0   0   Gall bladderMusculature 0   0 − 1+ Epithelia 1+ 1+ Lymphatic follicle 1+ 2+Musculature 2+ 1+ Ganglion cells 2+ 3+ Vascular endothelium 2+ 1+Vascular endothelium 1+ 1+ Cystic duct Small intestine Epithelium 3+ 3+Epithelia 2+ 3+ Musculature 2+ 1+ Musculature 1+ 1+ Ganglion cells 3+ 3+Stroma 1+ 2+ Vascular endothelium 2+ 2+ Ganglion cells 3+ 3+ TestisVascular endothelium 1+ 1+ Leydig cells 3+ 1+ Colon/Rectum Sertoli cells1+ − 2+ 1+ Epithelia 1+ 1+ Germ cells 1+ − 2+ 1+ Lymphatic follicles 1+1+ Vascular endothelium 1+ 1+ Plasma cells 1+ 1+ Rete testis Vascularendothelium 1+ 0   Epithelium 2+ 2+ Epididymis Placenta Epididymis duct2+ 2+ Chorionic epithelium 3+ 2+ Efferent ductulus 2+ 2+ Amnioticepithelium 2+ 2+ Stroma 1+ 1+ Decidual cells2+− 3+ 2+ Vascularendothelium 1+ 1+ Stroma cells 0   +/− Seminal gland Vascularendothelium 1+ 1+ Epithelium 2+ 3+ Fetal membranes Musculature 1+ 1+Amniotic epithelium 3+ 2+ Vascular endothelium 2+ 2+ Decidual cells 3+1 + Deferent duct Fibroblasts 3+ Epithelium 2+ 3+ Cervix uteriLongitudinal muscle 0   +/− Glandular epithelium 0   0   layer Annularmuscle layer 2+ 3+ Vascular endothelium 1+ 0 − 1 Vascular endothelium 2+3+ Stroma 1+ 0 − 1 Prostate Fallopian tube Glandular epithelium 2+ 2+Epithelium 2+ 3+ Musculature 1+ 1+ Musculature 0   1+ Vascularendothelium 1+ − 2+ 1+ − 2+ Vascular endothelium 1+ 2+ Kidney BreastTubules 2+ 1+ Epithelia mammary 2+ 2+ gland lobules Glomerules 0   0  Duct epithelium 2+ 2+ secretory ducts Medullary epithelium 1+ 1+Fibroblasts 0   1+ Vascular endothelium 0 − 1+ 0 − 1+ Plasma cells 2+2+Bladder Vascular endothelium 1+ 0   Urothelium 2+ 1+ − 2+ ThyroidMusculature 2+ 1+ Follicular epithelium 2+ Plasma cells 2+ 2+ Stroma 1+1+ Fibroblasts 1+ − 2+ 1+ − 2+ Vascular endothelium 1+ 0   Peripheralnerve 0   Thymus Adrenal gland Hassall's bodies 2+ − 3+ 2+ Glomerularzone 2+ 1+ Follicles 1+ 2+ Fascicular zone 1+ − 2+ (1+) Mantle zone (1+)(1+) Reticular zone 3+ (1+) Starry sky 1+ 1+ macrophages Medulla 0   0  Spleen ++ Vascular endothelium 1+ (1+) Tonsils +/− +/− Endometrium Lymphnodes +/− +/− Glandular epithelium 3+ 2+ Maxillary sinus Stroma cells0   1+ Respiratory epithelium 2+ 2+ Myometrium 1+ 1+ Plasma cells 3+ 3+Vascular endothelium 1+ 2+ Vascular endothelium 1+ 1+ Lung Fatty tissue2+ 2+ Bronchial epithelium 2+ 1+ Vascular endothelium 2+ 2+ Alveolarepithelium 1+ − 2+ 1+ Skin Bronchial glands 1+ 1+ Epidermis 2+ 1+ − 2+Cartilage 3+ 1+ Dermis (1+) 0   Musculature 1+ 1+ Hypodermis (1+) 0  Alveolar macrophages 2+ 2+ Sweat glands 1 + 0   Elastic fibers 2+ − 3+2+ − 3+ Vascular endothelium 1+ 0   Vascular endothelium 1+ 1+Endocardium 0   0   Skeletal muscles 2+ 1+ Fibroblasts 2+ − 3+ 2+ − 3+ 0= negative 1+ = weakly positive 2+ = moderately strong positive 3+ =strongly positive

Endocrine cells such as the islets of Langerhans of the pancreas, theLeydig cells of the testicular interstitium, the decidual cells of theplacenta, the oxyntic gland body of the stomach cardia, and the highlycylindrical epithelium of the cystic duct, display a strong reaction,which in part, shows fine granules. Strongly positive reactions wereobserved in plasma cells located in tissue structures and ganglioniccells and nerve cells of the cortex. The decidual cells, the amnioticepithelium and the fibroblasts of fetal membranes displayed very strongimmunohistological stainability, as did the epithelium lining theseminal glands and the enterocytes of the small intestine.

Studies of formalin-fixed, paraffin-embedded tumor material ofurological tumors displayed a weak to moderately strongintracytoplasmatical reaction of different differentiatedadenocarcinomas of the prostate. Tumor cells of seminomatous testiculartumors showed only a weak intracytoplasmatic reaction whilenon-seminomatous tumors (embryonic carcinomas and chorionic carcinomas)had a widely increased stainability of the tumor cells, indicatingincreased concentrations of FSAP.

The diagnostic method of the invention thus allows animmunohistochemical detection of pathological processes in a widevariety of organs.

EXAMPLE II BRUNECK STUDY Study Subjects

The Bruneck study is a prospective population study aimed at throwinglight on epidemiology and etiology of carotid atherosclerosis (1–6). Thestudy population was recruited in 1990 as a sample stratified accordingto sex and age and all the Bruneck inhabitants from 40 to 79 years ofage (125 women and 125 men from each of decades 5 to 8 of age, n=1000).In total, 93.6% took part, with 919 completing the data acquisition.During the follow-up period between summer 1990 and 1995(quinquennium₁—Q₁), a subgroup of 62 individuals died, while one subjectmoved house. Follow-up in the remaining population was 96.5% complete(n=826) (1–3). Before entry in the study, all the participants gavetheir consent after they had been informed about the study. As part ofthe follow-up in 1995, blood samples were taken to obtain DNA.Unsatisfactory PCR products were obtained in 16 cases, i.e. 810 men andwomen remained for the main analysis. Of these subjects, 94 died betweensummer 1995 and 2000 (quinquennium₂—Q₂). A total of 675 subjectsunderwent ultrasound investigation again in 2000 (follow-up rate amongthe survivors 94.3%) (6).

Clinical History and Examination

The study protocol included a clinical examination with priority forcardiological and neurological items and standardized questionnairesconcerning the current or past vulnerability due to potential vascularrisk factors (3–5). For smokers and former smokers, the average numberof cigarettes smoked each day, and the pack-years, were recorded. Thealcohol consumption was quantified as grams per day and classified infour categories (3). Systolic and diastolic blood pressures were themeans of three measurements, in each case measured after resting for ≧10minutes. Hypertension was defined as a blood pressure of ≧160/95 orintake of antihypertensive agents (WHO definition). A standardized oralglucose tolerance test was carried out on all subjects excepting thosepreviously known to be diabetic. Diabetes mellitus was entered aspresent for those subjects whose fasting blood glucose level was ≧140mg/dl (7.8 mmol) and/or who had a 2-hour level (oral glucose tolerancetest) of ≧200 mg/dl (11.0 mmol/l).

Laboratory Methods

After the subjects had taken no food and not smoked for at least 12hours, blood was taken from the antecubital vein (3–6). Totalcholesterol and cholesterol with high density lipoprotein weredetermined enzymatically (CHOD-PAP method, Merck, Darmstadt, Germany),and lipoprotein(a) concentrations were measured using an ELISA (Immuno,Vienna, Austria). The cholesterol with low density lipoprotein wascalculated from the Freidewald formula. Fibrinogen was measured by themethod of Clauss, and the antithrombin III using a chromogenic assay.The Leiden mutation of factor V was detected by allele-specific PCRamplification (3).

FSAP antigen concentrations and scuPA-activating effect were determinedas described recently (7, 8). Stated briefly, an ELISA with monoclonalantibodies (mAb) against FSAP was used for antigen quantification. Theactivity assay comprised an immunoadsorption onto microtiter platescoated with antibodies, a washing step and subsequent activation ofprourokinase by FSAP, which was quantified by photometric observation ofthe amidolysis of a chromogenic substrate for urokinase. Pooled plasmafrom more than 200 healthy blood donors was used as arbitrary standardfor both assays. A plasma equivalent unit (PEU) was defined as the FSAPantigenic activity present in one milliliter of the pooled plasma, whichcorresponds on average to 12 μg/ml (8).

DNA extraction and FSAP genotyping: high-quality DNA was obtained fromfrozen whole blood using a GenomicPrep Blood DNA Isolation Kit (AmershamPharmacia Biotech). Ten ml of extracted DNA were amplified in 100 μl of1×PCR standard reaction buffer with 50 pmol of the correspondingexon-specific forward and reverse primer, 1.5 mM MgCl₂, 0.2 mM dNTP and2.5 units of Taq DNA polymerase (Perkin Elmer, Langen, Germany); aninitial 2-minute denaturation at 94° C. was followed by 35 thermocycleseach for 30 seconds at 94° C., 30 seconds at 50° C. and 40 seconds at72° C., which was followed by a final elongation step at 72° C. for 5minutes. The pairs of primers used have recently been described inRoemisch J., Feussner A., Nerlich C., et al. The frequent Marburg Ipolymorphism impairs the prourokinase activating potency of the factorVII-activating protease. Blood Coag Fibrinol 2002; 13:1–9, which isincorporated herein by reference.

Scanning Protocol and Definition of the Ultrasound Endpoints

In the ultrasound examination, the internal carotid artery (bulbous anddistal sections) and common carotid artery (proximal and distalsections) on both sides were scanned using a 10 MHz probe and a 5 MHzdoppler (1,2). Atherosclerotic lesions were defined by two ultrasoundcriteria: 1) wall surface (protrusion into the lumen) and 2) walltexture (echogenicity). The maximum axial diameter of plaques wasdetermined in each of 16 vessel sections (intra-observation coefficientof variation 10% or 15% depending on the vessel section). The thicknessof the intima media was measured at the far walls of the common carotidartery (intra-observation coefficient of variation 7.9% (n=100)) (2).The scans were performed in 1990, 1995 and 2000 by the same experiencedultrasonic specialist, the clinical findings and laboratory values ofthe subjects being unknown to the ultrasonic specialist.

The development of atherosclerosis was characterized by the appearanceof new plaques in previously normal sections. Thresholds of 0.7 mm(common carotid artery) and 1.0 mm (internal carotid artery) wereintroduced as minimum requirements concerning the plaque diameters inthe definition of developing atherosclerosis, because smaller lesionswere difficult to distinguish from focal/diffuse wall thickenings (1).Progression of non-stenotic lesions was defined as a relativeenlargement of the plaque diameter of more than twice the measurementerror of the method (1). In the current analysis, both processes werecombined to a single result category referred to as “earlyatherogenesis” for easier presentation and because of the fact that mostof the described risk factors were common to these processes. An“advanced atherogenesis” was assumed whenever the criterion ofprogression was met and the lumen was narrowed by >40%. As describedelsewhere (1–5), the cutoff at 40% appeared to correspond to abiological threshold in our population, at which marked changes in thegrowth kinetics of plaques (continuous, slow and diffuse growth versusoccasional and focal expansions of prominent lesions), in the riskprofiles (conventional risk factors versus procoagulation risk factors)and in the process of vascular renewal (compensating or overcompensatingversus insufficient or even absent) occurred, indicating a switch in theunderlying pathogenetic mechanisms from conventional atherogenesis toatherothrombosis.

The reproducibility of the ultrasound categories was “nearly perfect”(kappa coefficients of >0.8, obtained from two independent measurementscarried out by the same ultrasound specialist in a reproducibilitysample of n=100 (1–3).

Statistical Analysis

Possible associations between FSAP mutations and the various stages ofatherogenesis were examined by means of logistic regression analysis. Abase model was adjusted only in relation to age and sex. Multivariateequations were fitted by a stepwise progressive selection procedure asalready described (p values for entry and exclusion 0.10 and 0.15respectively) (3, 10). Age and sex were additionally inserted into thesemodels in order to take account of the age and sex structure of thepopulation sample. The main analysis was concentrated on the periodbetween 1990 and 1995 (Q₁). Analysis of advanced atherogenesis wasrestricted to subjects already suffering from atherosclerosis at thestart of the study (n=326).

The regression-standardized atherogenesis risks were calculated for anumber of risk factors. The marginal method of the regression adjustmentprocedure was used, because it is not based on the rare-diseaseassumption (11).

Results

In the Bruneck study cohort (n=810), 36 subjects were heterozygous forthe Marburg I mutant of FSAP (17 men and 20 women) and one subject washomozygous, corresponding to an overall carrier rate [95% Cl] in thegeneral population of 4.4% [3.0% to 5.8%]. A cosegregation of theMarburg II mutant (E393Q) of FSAP was observed in 16 of the 37individuals (43 percent, 8 men and 8 women), while the Marburg I mutantoccurred in isolation in the remaining 21 subjects (57 percent, 9 menand 12 women).

Plasma samples from the subpopulation (n=82) were investigated for FSAPantigen concentrations and corresponding prourokinase-activatingeffects. In 76 subjects with wild-type FSAP, the average (±2×SD) antigenconcentrations, activity concentrations and activity/antigen ratios wererespectively 0.991 (0.552 to 1.430) PEU/ml, 1.036 (0.614 to 1.458)PEU/ml and 1.07 (0.63 to 1.51). In contrast thereto, all six carriers ofthe Marburg I mutant in this subgroup showed a distinctly reduced invitro capacity to activate prourokinase (<0.150 to 0.626) andactivity/antigen ratios of 0.38 to 0.58. There was hardly any overlap inthe distribution of these parameters in the two genetic groups.

During the five-year follow-up period between 1990 and 1995 (Q₁), atotal of 384 of the 810 subjects in the study (47.4%) developed newatherosclerotic lesions or showed extension of nonstenotic lesions(early atherogenesis), and 92 of 326 individuals (28.2%) withpre-existing plaques showed stenotic transformations (advancedatherogenesis). As expected, no relation was found between Marburg Imutant and early atherogenesis (age/sex adjusted, multivariate oddsratios [95% Cl] of 0.6 [0.3 to 1.4] and 0.7 [0.3 to 1.7]. Consistentwith this, there were no differences in the thickness of the intimamedia of the common carotid artery between the carriers of wild-typeFSAP (0.95 mm) and of the Marburg I mutant of FSAP (0.94 mm; P=0.853 forthe difference). However, it emerged that the mutant is a strong riskfactor for the advanced putative atherothrombotic stage in atherogenesis(age/sex adjusted odds ratio [95% Cl] 3.5 [1.1 to 11.4], P=0.036). Theassociation remained statistically significant on adjustment of thelogistic regression model for other relevant risk factors (tab. 1). Therisk profile for advanced stenotic atherosclerosis also includeddiabetes, a high fibrinogen concentration, a low antithrombinconcentration, a high platelet count, smoking, alcohol consumption(small amounts protective), Lp(a)>0.32 g/l and Leiden mutation of factorV. There were no sex-specific differences in the risk factors, and noevidence of differential effects of the Marburg I mutant was found insubpopulations arranged according to age, level of risk and life style.Exclusion of subjects taking aspirin, antihypertensive agents,antidiabetics or lipid-lowering agents likewise did not affect theresults. Regression-standardized risks of advanced atherogenesis for anumber of major risk factors (Marburg I mutant, IGT/diabetes, highlipoprotein(a) concentration, smoking, factor V mutation, highfibrinogen concentration and low antithrombin concentration) are shownin table 2. Subjects with none of the risk factors had a low risk fordevelopment/progression of carotid stenosis, whereas subjects with acluster of more than two factors almost obligatorily experiencedadvanced atherogenesis.

The Marburg II mutant had no effect on in vitro activation ofsingle-chain plasminogen activators by FSAP. Accordingly, it was notunexpected that no association between this mutation and atherogenesiscould be found in our analyses. On comparison of subjects with wild-typeFSAP and carriers of the Marburg II mutant, of the Marburg I mutant andcarriers of both genetic deviations, the multivariate odds ratios [95%Cl] for advanced atherosclerosis were 1.6 [02 to 13.7], P=0.669, 6.2[1.1 to 36.0], P=0.048 and 7.1 [1.1 to 45.1], P=0.037.

To demonstrate that our findings are also consistent over longerperiods, the calculations were repeated with the data from the ten-yearfollow-up period between 1990 and 2000 (Q₁₊₂). In these equations, themultivariate relation between the Marburg I mutant of FSAP and advancedatherosclerosis (same adjustments as for the original analysis) wasagain statistically significant (multivariate odds ratio [95% Cl] 4.1[1.1 to 14.8], P=0.045).

TABLE 5 Multivariate logistic regression analysis of advancedatherogenesis according to age, sex, Marburg I mutant of FSAP and otherpotential vascular risk factors. Means ± standard deviation (%) AS −AS + Odds ratio Variable (n = 234) (n = 92) (95% CI) P value Step Age, y64.9 ± 9.2 67.8 ± 8.0 1.87(1.19 − 2.92) .0064 0 Female sex 109(46.6%)32(34.8%) 0.56(0.25 − 1.25) .1555 0 Glucose tolerance <.0001 1 IGT 20(8.5%) 16(17.4%) 3.31(1.37 − 7.99) .0081 DM  10(8.1%) 21(22.8%)6.38(2.71 − 14.99) <.0001 Cigarettes/day 3.2 ± 7.2 6.6 ± 9.6 1.77(1.30 −2.40) .0003 2 Lp(a) > 0.32 g/l  36(15.4%) 25(27.2%) 4.06(1.83 − 8.96).0005 3 Alcohol consumption .0043 4    <1 g/d 114(48.7%) 42(45.6%) 1.00 1–50 g/d  60(25.7%) 15(16.3%) 0.26(0.10 − 0.66) .0046 51–99 g/d 37(15.8%) 17(18.5%) 1.03(0.40 − 2.70) .9475  ≧100 g/d  23(9.8%)18(19.6%) 1.90(0.63 − 5.69) .2535 Fibrinogen, g/l 2.7 ± 0.6 2.9 ± 0.61.53(1.12 − 2.09) .0083 5 Marburg I FSAP  5(2.1%)  8(8.7%) 6.63(1.58 −27.72) .0099 6 mutation Factor V mutation  5(2.1%)  7(7.6%) 4.70(1.19 −18.55) .0291 7 Antithrombin III, % 96.3 ± 13.0 92.8 ± 16.4 0.74(0.55 −1.00) .0500 8 Platelet count, × 10⁹/l 217.4 ± 56.5  230.3 ± 56.6 1.32(0.98 − 1.77) .0769 9

Odds ratios (OR), 95% confidence interval (95% Cl) and p values (P) werederived from the logistic regression analysis of advancedatherosclerosis (development/progression of stenotic carotidatherosclerosis) in relation to age, sex and vascular risk factors. Themodel was fitted by a stepwise progressive selection process (step . . .entry step). The ORs were calculated for a 1-SD unit change of givenvariables.

AS−: group without advanced atherogenesis, AS+: group with advancedatherogenesis. This analysis was concentrated on the 326 subjects whoalready suffered from atherosclerosis at the start of the study in 1990.

REFERENCES

1. Kiechl S, Willeit J. The natural course of atherosclerosis. Part I:incidence and progression. Arterioscler Thromb Vasc Biol 1999; 19:1480–90.

2. Kiechl S, Willeit J. The natural course of atherosclerosis. Part II:vascular remodeling. Arterioscler Thromb Vasc Biol 1999; 19: 1491–8.

3. Willeit J, Kiechl S, Oberhollenzer F, et al. Distinct risk profilesof early and advanced atherosclerosis. Prospective results from theBruneck Study. Arterioscler Thromb Vasc Biol 2000; 20: 529–37.

4. Kiechl S, Egger G, Mayr M, et al. Chronic infections and the risk ofcarotid atherosclerosis. Prospective results from a large populationstudy. Circulation 2001; 103: 1064–70.

5. Kiechl S, Willeit J, Egger G, Poewe W, Oberhollenzer F. Body ironstores and the risk of carotid atherosclerosis. Prospective results fromthe Bruneck Study. Circulation 1997; 96: 3300–7.

6. Kiechl S, Lorenz E, Reindl M, Wiedermann C J, Oberhollenzer F, BonoraE, Willeit J, Schwartz D A. Toll-like receptor 4 polymorphisms andatherogenesis in humans. N Engl J Med 2002; 347: 185–92.

7. Roemisch J, Feussner A, Stohr H A. Quantification of the factor VII-and single-chain plasminogen activator-activating protease in plasmas ofhealthy subjects. Blood Coagul. Fibrinolysis. 2001; 12: 375–83.

8. Kannemeier C, Feussner A, Stohr H A, Weisse J, Preissner K T,Roemisch J. Factor VII and single-chain plasminogen activator-activatingprotease: activation and autoactivation of the proenzyme. Eur J Biochem.2001; 268: 3789–96.

9. Roemisch J, Feussner A, Nerlich C, Stoehr H A, Weimer T. The frequentMarburg I polymorphism impairs the prourokinase activating potency ofthe factor VII-activating protease (FSAP). Blood Coag Fibrinol 2002; 13:1–9.

10. Hosmer D W, Lemeshow S. Applied Logistic Regression. New York: JohnWiley & Sons, 1988.

11. Wilcosky T C, Chambless L E. A comparison of direct adjustment andregression adjustment of epidemiological measures. J. Chron Dis 1985;38: 849–56.

Figure Legend

FIG. 1 shows the regression-adjusted risk of advanced atherogenesis as afunction of the vascular risk factors present in an individual (MarburgI mutant of factor VII-activating protease, IGT/diabetes, lipoprotein(a)concentration >0.32 g/l, smoking, Leiden mutation of factor V,fibrinogen concentration (Q₅, >3.2 g/l) and antithrombin concentration(Q₁, <84%)).

1. An isolated coagulation factor VII-activating protease (FSAP) mutantderived from a human donor, which is an atherothrombosis risk factor,and which comprises a Gly to Glu exchange at amino acid position 534 ofthe FSAP proenzyme sequence, said amino acid sequence position definedaccording to the proenzyme amino acid sequence of SEQ ID NO:5.
 2. Therisk factor as claimed in claim 1, wherein the FSAP mutant furthercomprises a Glu to Gin exchange at amino acid position 393 of theproenzyme sequence, said amino acid sequence position defined accordingto the proenzyme amino acid sequence of SEQ ID NO:5.
 3. The risk factoras claimed in claim 1, wherein the FSAP mutant is encoded by a proenzymenucleotide sequence comprising a G to A base exchange at position 1601,said nucleotide sequence position defined according to the proenzymenucleotide sequence of SEQ ID NO:1.
 4. The risk factor as claimed inclaim 3, wherein the FSAP mutant is encoded by a proenzyme nucleotidesequence further comprising a G to C base exchange at position 1177,said nucleotide sequence position defined according to the proenzymenucleotide sequence of SEQ ID NO:1.
 5. The risk factor as claimed inclaim 1, wherein the FSAP mutant has partially or completely lost theability to activate single-chain plasminogen activators, as comparedwith wild-type FSAP.
 6. The risk factor as claimed in claim 1, whereinthe FSAP mutant has partially or completely lost the ability to activateprourokinase, as compared with wild-type FSAP.
 7. The risk factor asclaimed in claim 1, which indicates a genetic predisposition to thedevelopment of atherosclerotic disorders and their sequelae, orthrombotic disorders and their sequelae.
 8. The risk factor as claimedin claim 7, which indicates a genetic predisposition to the developmentof at least one of arterial and venous occlusive disorders.
 9. The riskfactor as claimed in claim 7, which indicates a genetic predispositionto the development of at least one of atherosclerotic and thromboticrestrictions of organ functions.
 10. The risk factor as claimed in claim7, which indicates a genetic predisposition to the development of one ormore of angina pectoris, myocardial infarction, and strokes.
 11. Theatherothrombosis risk factor as claimed in claim 1, wherein the abilityto activate single-chain plasminogen activators, measured in at leastone of whole blood and blood plasma, is reduced as compared withwild-type FSAP.
 12. The atherothrombosis risk factor as claimed in claim1, wherein the ability to activate prourokinase, measured in at leastone of whole blood and blood plasma, is reduced as compared withwild-type FSAP.
 13. A diagnostic method for identifying theatherothrombosis risk factor as claimed in claim 1, which comprises: (a)determining FSAP prourokinase activating activity in one or more bodyfluids of an individual; (b) comparing said FSAP prourokinase activatingactivity in one or more body fluids of an individual to FSAPprourokinase activating activity in a standard comprising wild-typeFSAP; (c) identifying a reduced FSAP prourokinase activating activity insaid one or more body fluids of the individual compared to the FSAPprourokinase activating activity in said standard; and (d) analyzing atleast one of genomic DNA, mRNA, or cDNA of the individual with thereduced FSAP prourokinase activating activity to detect the presence ofa heterozygous or homozygous mutation in the FSAP nucleotide sequence,said mutation comprising a G to A base exchange at nucleotide position1601, said nucleotide sequence position defined according to theproenzyme nucleotide wherein the presence of said mutation identifiesthe atherothrombosis risk factor sequence of SEQ ID NO:1.
 14. Thediagnostic method as claimed in claim 13, further comprising determiningthe FSAP protein concentration in said one or more body fluids of theindividual, and calculating the ratio between said FSAP proteinconcentration and FSAP prourokinase activating activity in said one ormore body fluids.
 15. The diagnostic method as claimed in claim 13,wherein the one or more body fluids comprise blood plasma.
 16. Thediagnostic method as claimed in claim 13, further comprising determiningthe activation of single-chain tissue plasminogen activators in one ormore body fluids of the individual.
 17. The diagnostic method as claimedin claim 13, further comprising analyzing at least one of the genomicDNA, mRNA, or cDNA of the individual to detect the presence of aheterozygous or homozygous mutation in the FSAP nucleotide sequence,said mutation comprising a G to C base exchange at nucleotide position1177, said nucleotide sequence position defined according to theproenzyme nucleotide sequence of SEQ ID NO:1.
 18. The diagnostic methodas claimed in claim 13, wherein the FSAP prourokinase activatingactivity is measured by: (a) incubating said one or more body fluids ofthe individual on a solid support to immobilize FSAP on said solidsupport; (b) washing the support; and (c) incubating the FSAPimmobilized on the support with reagents which allow determination ofthe prourokinase activating activity of the FSAP immobilized on thesupport.
 19. The risk factor as claimed in claim 3, wherein the FSAPmutant is encoded by a proenzyme nucleotide sequence further comprisinga base exchange at position 183 which does not lead to an amino acidexchange, said nucleotide sequence position defined according to theproenzyme nucleotide sequence of SEQ ID NO:1.
 20. The risk factor asclaimed in claim 3, wherein the FSAP mutant is encoded by a proenzymenucleotide sequence further comprising a base exchange at position 957which does not lead to an amino acid exchange, said nucleotide sequenceposition defined according to the proenzyme nucleotide sequence of SEQID NO:1.